Plasma preparation



PLASMA PREPARATION Heron 0. Singher, Plainfield, and Emanuel A. Swart, Somerville, N. J., assignors to Ortho Pharmaceutical Corporation, a corporation of New Jersey No Drawing. Application May 27, 1954 Serial No. 432,896

4 Claims. (Cl. 167-74) This invention relates to a plasma preparation for use as a standard in the determination of the prothrombin time of a thromboplastic material. More particularly, this invention relates to a plasma preparation obtained by mixing oxalated bovine or equine plasma, with a plasma fraction obtained from prothrombin-free rabbit, bovine, human or equine plasma.

Thromboplastic material obtained from rabbit brain or lung tissue, or mixtures thereof, or from other sources varies in activity with each preparation, and each preparation must be standardized in order that prothrombin time measurements of dififerent human blood specimens may be correlated. Heretofore, preparations having thromboplastic activity have been standardized by the use of rash, normal, oxalated, human plasma; however, the prothrombin time of human plasma specimens may vary significantly.

It is an object of this invention to provide a plasma preparation suitable for use as a standard in the determination of the thromboplastic activity of thromboplastic material.

Other objects and advantages of the invention Will be apparent from the following descriptions and exemplary disclosures. V

The objects of this invention are accomplished by plasma preparation prepared by the addition to oxalated bovine or equine plasma of a prothrombin-free plasma extract obtained by the extraction with a buffered aqueous alcohol solution of prothrombin-free rabbit, bovine, human or equine plasma from which albumin and alphaglobulinsthave been substantially removed by extraction with a first buffered aqueous alcohol solution.

More particularly, in preparing the standard plasma preparation of this invention, the horse, bovine, human or rabbit plasma fraction, and preferably a fraction derived from horse plasma, is dissolved in oxalated bovine plasma in an amount such that its concentration with respect to the bovine plasma is about two to ten milligrams per milliliter, and preferably three milligrams per milliliter. If the concentration of plasma fraction with respect to bovine plasma is less than about two and greater than about ten milligrams per milliliter, the prothrombin time obtained with the standard plasma prepara tion is significantly different from the time obtained with normal, oxalated, human plasma, when both are used in the determination of the prothrombin time of the same thromboplastic material. One milliliter portions are lyophilized to provide a stable, solid material which, upon the addition of one milliliter of distilled water, provides 1 suspension having the same prothrombin time as the }-.verage, normal, fresh, oxalated human plasma when letermined by our modification of the Shapiro-Weiner nethod.

The bovine plasma is prepared by centrifuging oxalated bovine blood containing percent by volume of 0.1 molar sodium oxalate solution. The supernatant from the centrifugation is diluted with 0.85 ,percent sodium chloride solution such'that the bovine plasma is present nited States Patent in the diluted solution in an amount of 60 to percent by volume, and preferably 70 percent by volume. Dilution in this range results in a clot very similar in appearance to a clot formed in human blood. Undiluted oxalated bovine plasma may also be used.

In the preparation of the prothrombin-free horse, bovine, human or rabbit plasma extract for addition to oxalated bovine or equine plasma to provide the standard plasma preparation of this invention, horse, bovine, hu man or rabbit blood to which a heparin sodium solution has been added to prevent coagulation, is centrifuged in order to obtain the plasma. The plasma .is slurried with two to five percent, and preferably five percenuof barium sulfate, the barium sulfate being expressed in grams, and the plasma in cubic centimeters. At least two percent of barium sulfate is required to remove all the prothrombin, and the presence of more than five percent results in a mixture of such a consistency that physical handling thereof is extremely difiicult. The slurry is stirred for one hour in order that the maximum amount of prothrombin be completely adsorbed, and is then centrifuged. It is preferred that the adsorption be repeated to insure complete removal of prothrombin, preferably with the same amount of barium sulfate, and upon centrifugation after the second adsorption, the plasma is pro'thrombin free. Albumin and alpha-globulins are extracted from the prothrombin-free plasma by the addition of a first buffered aqueous alcohol solution and centrifugation. The alcohol may be ethanol, methanol, or a mixture of methanol and ethanol and may be present in an amount of from about 15 to about 30 percent by volume, but it is preferred that the amount be about 25 percent. if the amount is less than about 15 percent, a significant amount of inactive substances remain in the precipitate, and if the amount is greater than about 30 percent a substantial amount of the desired plasma fraction is present in the solution. The first aqueous alcohol solution is buttered within the pH range of from 4 to 5, the preferred pI-l being 4.2 to 4.6. Buffer-- ing below a pH of 4 results in incomplete solution of albumin in the extracting solution and buffering above a pH of 5 results in a significant amount of the desired plasma fraction being lost in the extracting solution. in general, any buffer system capable of maintaining the pH of the mixture of the first aqueous alcohol solution and prothrombin-free plasma within the range of 4 to 5 may be used. The preferred buffer system is sodium acetateacetic acid, however, buffer systems such as sodium succinate-succinic acid, and sodium acid phthalate-phthalaic acid, have been found suitable.

The volume of first aqueous alcohol solution usedmay vary widely, but the most efficient removal of albumin and alpha-globulin from the prothrombin-free plasma is accomplished when the volume is three to five times the volume of the plasma. It is preferred that the volume of the first aqueous alcohol solution be about four times the volume of the plasma.

It is necessary that the extraction and removal by cen-v trifugation of albumin and alpha-globulins in solution in the first aqueous alcohol solution be accomplished at a low temperature in order that they be efficiently removed from the plasma. Extraction and removal at a higher temperature results in denaturation of albumin and alpha-globulins by the alcohol and incomplete removal thereof in the extracting solution. The prothrombin-free horse, bovine, human or rabbit plasma is cooled to 5 to 0 C., and preferably to 0 C.,,and the first aqueous alcohol solution, which has been cooled to 5 to 10 C., is slowly added with stirring and during the course of the addition, the temperature of the mixture is maintained between 0 and 5 C., and preferably at about 5 C. After addition is completed the mixture is stirred for about 30 minutes and during this time the temperature of the m xture is maintained at to C., and preferably at -5 C. Immediately after stirring is discontinued, the mixture is centrifuged and during the 'centrifugation the temperature is maintained at 0 to -5 C., and preferably 5 C. The supernatant, which consists mainly of albumin and-alpha-globulins is discarded and the residue is extracted with a second buffered aqueous alcohol solution to obtain the desired plasma fraction.

The alcohol in the second aqueous alcohol solution may be ethanol, methanol, or a mixture of ethanol and methanol and the alcohol, or mixture of alcohols, is present in an amount from 5 to percent by volume and preferably in an amount of about 16 percent by volume. The second aqueous alcohol solution is buttered at a pH within the range of from 6 to 8, and preferably 6.8 to 7.2. A solution buffered outside this pH range extracts significantly less of the desired plasma fraction from the residue of the first extraction. The second aqueous alcohol solution contains alkali metal salt of an amino acid having not more than six carbon atoms, such as alanine, glycine, proline, or serine. In general, other amino acids are not sufiiciently soluble in the second extracting solution. The alkali metal salt acts as a stabilizing agent for the material to be extracted from the plasma, as a solubilizer for betaand gamma-globulins, and as an active part of the buffer system. The amino acid is present in an amount within the range of 4 to 6 percent by weight, and preferably 5.4 to 5.8 percent by weight. At a concentration less than 4 percent by weight, the solubilizing effect of the amino acid is significantly decreased; on the other hand, the solubilizing effect is not increased when the concentration is greater than 6 percent. Phosphates such as monoor di-sodium phosphate are also present in the second aqueous alcohol solution as part of the buffer system and the pH of the solution is adjusted to the desired value with an alkali metal hydroxide. Any buffer system may be used which is ef fective within a pH range of 6 to 8.

The amount of the second aqueous alcohol solution used in the extraction of the albumin and alpha-globulinfree residue may vary widely but an amount of solutionthree to five times, and preferably four times, the volume of the original plasma results in the most eflicient recovery of the desired plasma fraction. The mixture of residue and second aqueous alcohol solution is stirred at a temperature of 0 to 5 C., and preferably at -5 C., for about 30 minutes, and then centrifuged and the residue from the centrifugation which consists mainly of beta-globulins and fibrinogen is discarded. The supernatant, which contains the desired plasma fraction, is filtered, dialyzed against distilled water at a temperature of 1 to 5 C., and the dialysate is lyophilized. (The term dialysate as used in this specification designates the material which has failed to pass through the dialysis membrane.) Denaturation of the desired plasma fraction is at a minimum when the temperature during dialysis is within the range of 1 to 5 C. The solid material obtained upon addition to thromboplastic material enhances its thromboplastic activity.

In order that those skilled in the art may better un derstand how the present invention may be carried into effect, the following examples are given by way of illustration and not by way of limitation.

Example I In the preparation of the prothrombin-free plasma extract from horse blood, fifty ml. of heparin sodium solution were added to 20 liters of horse blood and the mixture was centrifuged for 30 minutes at C. Nine liters of the supernatant plasma were slurried with 450 grams of barium sulfate at 25 C., stirred for one hour and centrifuged. The prothrombin-free plasma obtained,

which. had a volume of eight and one-half liters, was cooled to 0 C. Thirty-four liters of an aqueous alcohol solution buffered at a pH of 4.6 was cooled to a temperature of 0 C. and added to the plasma at such a rate that the temperature of the mixture throughout the addition was below 0 C. The mixture was stirred for 30 minutes at 5 C. after addition was complete and then centrifuged and the temperature of the mixture was maintained at a temperature of 5 during the centrifugation. The supernatant, which contained substantially all the albumin and alpha-globulins of the plasma, was discarded. The aqueous alcohol solution containe per liter, 12 /2 ml. of methanol, 237 /2 ml. of 95 percent ethanol, and 0.6 ml. of an acetate buffer solution prepared by diluting a mixture of 20 ml. of 4 molar so dium acetate and 40 ml. of 10 molar acetic acid to 100 ml. with water. The residue from the centrifugation was finely dispersed with a spatula in 34 liters of the aqueous alcohol extracting solution buffered at a pH of 7. The temperature of the extracting solution and residue during the dispersion was maintained at 5 C. and after dispersion was complete the mixture was stirred for 30 minutes at a temperature of 5 C. and centrifuged. The temperature of the mixture during the centrifugation was maintained at 5 C. The supernatant was filtered and dialyzed against distilled water. The temperature of the supernatant during filtration and dialysis was maintained at 5 C. The dialysate was lyophilized and 350 grams of solid were obtained. The solid had no demonstrable thromboplastic activity. The aqueous alcohol extracting solution contained, per liter, 8 ml. of methanol, 152 ml. of 95 percent ethanol, 56 grams of glycine, 3.12 ml. of a solution of sodium glycinate, prepared by dissolving 4.5 grams of glycine and 2.0 grams of sodium hydroxide in 100 ml. of water, 4 ml. of 0.5 molar disodium hydrogen phosphate, and 2.76 ml. of 0.5 molar monosodium dihydrogen phosphate.

Nine hundred milligrams of the fraction obtained from horse blood were added to 210 ml. of oxalated bovine plasma which had been diluted with m1. of 0.85 percent sodium chloride solution, and the mixture was lyophilized. An amount of the lyophilized material corresponding to of the total amount was dissolved in 1. ml. of distilled water to provide a reconstituted standard plasma.

Example II In the preparation of the prothrombin-free plasma extract from rabbit blood, three ml. of heparin sodium solution were added to 1200 ml. of rabbit blood and the mixture was centrifuged for 30 minutes at 25 C. Six hundred twenty ml. of the supernatant plasma were slurried with 31 grams of barium sulfate at 25 C., stirred for one hour and centrifuged. The supernatant was slurried with 31 grams of barium sulfate, stirred for one hour at 25 C., and centrifuged. The prothrombin-free plasma obtained, which had a volume of 565 ml. was cooled to 0 C. Two thousand two hundred sixty ml. of an aqueous alcohol solution buffered at a pH of 4.6 was cooled to a temperature of 0 C. and added to the plasma at such a rate that the temperature of the mixture throughout the addition was below 0 C. The mixture was stirred for 30 minutes at -5 C. after addition was complete and then centrifuged and the temperature of the mixture was main tained at a temperature of -5 during the centrifugation The supernatant, which contained substantially all th albumin and alpha-globulins of the plasma, was discarde The aqueous alcohol solution contained per liter, 12 /2 m of methanol, 237 /2 ml. of percent ethanol, and 0.6 m of an acetate buffer solution prepared by diluting a mi ture of 20 ml. of 4 molar sodium acetate and 40 ml. 0 10 molar acetic acid to ml. with water. The residu from the centrifugation was finely dispersed with a spatul Y in 2260 ml. of the aqueous alcohol extracting solution buf fered at a pH of 7. The temperature of the extracting solution and residue during the dispersion was maintained at -5 C. and after dispersion was complete the mixture was stirred for 30 minutes at a temperature of 5 C. and centrifuged. The temperature of the mixture during the centrifugation was maintained at 5 C. The supernatant was filtered and dialyzed against distilled water. The temperature of the supernatant during filtration and dialysis was maintained at 5 C. The dialysate was lyophilized and eight grams of solid were obtained. The solid had no demonstrable thromboplastic activity. The aqueous alcohol extracting solution contained, per liter, 8 ml. of methanol, 152 ml. of 95 percent ethanol, 56 grams of glycine, 3.12 ml. of a solution of sodium glycinate, prepared by dissolving 4.5 grams of glycine and 2.0 grams of sodium hydroxide in 100 ml. of water, 4 ml. of 0.5 molar disodium hydrogen phosphate, and 2.76 ml. of 0.5 molar monosodium dihydrogen phosphate.

One thousand milligrams of the plasma fraction obtained from rabbit blood were dissolved in 100 milliliters of undiluted, oxalated, bovine plasma. One milliliter portions of the mixture were placed in glass vials and lyophilized. The lyophilized material, in a vial, was reconstituted before use as a standard by the addition thereto of one milliliter of distilled water.

Plasma preparations prepared according to Examples I and II may be used as a standard for the determination of the prothrombin time of thromboplastic materials in place of fresh, normal, oxalated, human plasma and the prothrombintime of a given thromboplastic material is substantially the same with the standard preparation as with fresh, normal, oxalated human plasma.

A thromboplastic material for use in thromboplastic activity determinations using the standard plasma preparation and fresh, normal, oxalated, human plasma was prepared as follows:

Seventy-six grams of frozen rabbit brain and 1440 grams of frozen rabbit lung were homogenized at 5 C. for one minute in the presence of 7600 m1. of an aqueous solution containing, per liter, 150 ml. of an alcoholic solution prepared by adding 7.5 ml. of methanol, 15 grams of glycine, 4.8 ml. of one molar aqueous sodium acetate solution, and 2.6 ml. of one molar aqueous acetic acid solution to 142.5 ml. of 95 percent ethanol. The homogenate was stirred for two hours at 5 C. and centrifuged at 5 C. for thirty minutes. The supernatant liquid was filtered, dialyzed against distilled water at 5 C. and lyophilized. Forty-four and forty-six hundredths grams of thromboplastic solid material were obtained.

The thromboplastic activity, as measured by the prothrombin time, of the thromboplastic material prepared as above was determined as follows:

A calcium-thromboplastin suspension was prepared in a test tube by adding 25 milligrams of lyophilized solid to 5 ml. of 0.85 percent aqueous sodium chloride solution, admixing by inverting the tube three or four times until a uniform suspension was obtained, keeping the suspension in a water bath at 465 0 C. for twenty minutes, centrifuging, cooling the supernatant to room temperature, adding 0.1 ml, of 0.25 molar calcium chloride solution to 4 inl. of the suspension, mixing as above, and centrifuging again. Two-tenths ml. of the slightly turbid supernatant liquid was added to 0.1 ml. each of the reconstituted standard plasmas prepared according to Examples I and H and to 0.1 ml. of fresh, oxalated human plasma which had been prepared by the addition of 0.1 molar aqueous sodium oxalate solution to fresh human *blood in the proportion of one part sodium oxalate solution to nine parts of blood and centrifugation of the oxalated blood. The mixtures were agitated at 37 C. by jtilting the test tube back and forth and timing the first j'appearance of a fibrin clot. Clot formation was detected in 16.8 and 16.5 seconds, respectively, after addition of the supernatant liquid to the standard plasmas of Ex- 6 amples I and II and in seventeen seconds after addition of the supernatant liquid to human plasma.

All determinations of thromboplastic activity, as measured by prothrombin time, were determined by our modification of the Shapiro-Weiner method for determining prothrombin time of blood, which is described in a book entitled: Coagulation, Thrombosis and Dionmarol, by Shapiro and Weiner, published in 1949 by the Brooklyn Medical Press, Brooklyn, New York.

It will be obvious to those skilled in the art that various changes may be made without departing from the spirit of the invention and therefore it is to be understood that the invention is not limited to what is described in the specification and examples but only as indicated in the appended claims.

What is claimed is:

l. A process for the preparation of a plasma fraction for use in the determination of the prothrombin time of thromboplastic material comprising the steps of: dissolving in oxalated bovine plasma two to ten milligrams per milliliter of oxalated plasma of a plasma fraction prepared from prothrombin-free plasma, selected from the class consisting of horse, bovine, human, and rabbit prothrombin-free plasma, by a process conducted at a temperature within the range of 5 to 0 C. throughout comprising adding thereto three to five volumes of a first aqueous alcohol solution buffered at a pH of 45 and containing about 15-30 percent by volume of an alcohol selected from the class consisting of methanol and ethanol and mixtures thereof, stirring and centrifuging the mixture, separating the supernatant from the residue; and adding to the residue three to five volumes of a second aqueous alcohol solution buffered at a pH of 68 and containing 5-20 percent by volume of an alcohol selected from the class consisting of methanol and ethanol and mixtures thereof and 4-6 percent by weight of an alkali meta-l salt of an amino acid having not more than six carbon atoms, stirring and centrifuging the mixture, filtering the supernatant, dialyzing the filtered supernatant against distilled water, and lyophilizing the dialyzed supernatant.

2. A process for the preparation of a plasma preparation for use in the determination of the prothrombin time of thromboplastic material comprising the steps of dissolving in oxalated bovine plasma diluted with an amount of 0.85 percent aqueous sodium chloride solution such that 60-90 percent by volume of the plasma is present in the diluted solution, two to ten milligrams per milliliter of diluted bovine plasma of a plasma fraction prepared from prothrombin-free plasma at a temperature of 5 to 0 C., selected from the class consisting of horse, bovine, human, and rabbit prothrombin-free plasma, by adding thereto at a temperature of 5 to 10 C., about four volumes of a first aqueous alcohol solution bufiered at a pH of 4.2-4.6 and containing about 25 percent by volume of an alcohol selected from the class consisting of methanol and ethanol and mixtures thereof, stirring and centrifuging the mixture, and separating the supernatant from the residue while the temperature is maintained at 0 to 5 C. and adding at a temperature of 0 to 5 C. to the residue at a temperature of 0 to 5 C., three to five volumes of a second aqueous alcohol solution buffered at a pH of 6.8 to 7.2 and containing about 16 percent by volume of an alcohol selected from the class consisting of methanol and ethanol and mixtures thereof and 5.4-5.8 percent by weight of an alkali metal salt of an amino acid having not more than six carbon atoms, stirring the mixture while the temperature is at 0 to 5 C., centrifuging the mixture, filtering the supernatant, and dialyzing the filtered supernatant against distilled water while the temperature is at 1 to 5 C., and lyophilizing the dialyzed supernatant.

3. A plasma fraction for use in the determination of the prothrombin time of thromboplastic material prepared by the steps of: dissolving in oxalated bovine plasma two to ten milligrams per milliliter of oxalated plasma of a plasma fraction prepared from prothrombinfree plasma, selected from the class consisting of horse, bovine, human, and rabbit prothrombin-free plasma, by a process conducted at a temperature Within the range of 5 to C. throughout comprising adding thereto three to five volumes of a first aqueous alcohol solution bufiered at a pH of 4-5 and containing about 15-30 percent -by volume of an alcohol selected from the class consisting of methanol and ethanol and mixtures thereof, stirring and centrifuging the mixture, separating the supernatant from the residue; and adding to the residue three to five volumes of a second aqueous alcohol solution buttered at a pH of 6-8 and containing -20 percent by volume of an alcohol selected from the class consisting of methanol and ethanol and mixtures thereof and 4-6 percent by Weight of an alkali metal salt of an amino acid having not more than six carbon atoms, stirring and centrifuging the mixture, filtering the supernatant, dialyzing the filtered supernatant against distilled water, and lyophilizing the dialyzed supernatant.

4. A plasma fraction for use in the determination of the prothrombin time of thromboplastic material prepared by the steps of: dissolving in oxalated bovine plasma diluted with an amount of 0.85 percent aqueous sodium chloride solution such that 60-90 percent by volume of the plasma is present in the diluted solution, two to ten milligrams per milliliter of diluted bovine plasma of a plasma fraction prepared from prothrombin-free plasma at a temperature of 5 to 0 C., selected from the class consisting of horse, bovine, human, and rabbit prothrombin-free plasma, by adding thereto at a temperature of 5 to 10 C., about four volume of a first aqueous alcohol solution buffered at a pH of 4.2-4.6 and containing about 25 percent by volume of an alcohol selected from the class consisting of methanol and ethanol and mixtures thereof, stirring and centrifuging the mixture, and separating the supernatant from "the residue while the temperature is maintained at 0 to 5 C. and adding at a temperature of 0 to -5 C. to the residue at a temperature of 0 to 5 C., three to five volumes of a second aqueous alcohol solution butfered at a pH of 6.8 to 7.2 and containing about 16 percent by volume of an alcohol selected from the class consisting of methanol and ethanol and mixtures thereof and 5.4-5.8 percent by weight of an alkali metal salt of an amino acid having not more than six carbon atoms, stirring the mixture while the temperature is at 0 to 5 C., centrifuging the mixture, filtering the supernatant, and dialyzing the filtered supernatant against distilled water While the temperature is at 1 to 5 C., and lyophilizing the dialyzed supernatant.

References Cited in the file of this patent Wintrobe: Clin. Hematology, 2nd ed., 1946, Lea and Febiger, Philadelphia, Pa., pp. 207-209.

Surgenor: J. A. C. S., vol. 74, No. 13, July 5, 1952, pp. 3448-3450.

Cohn: Ann. Int. Med, vol 26, No. 3, March 1947, pp. 341-352 (pp. 346, 347, 349-352 relied on).

Edsfll: Advances in Protein Chem., vol. 3, 1947, Academic Press Inc., N. Y. C., pp. 440-445.

Callaham: Chem. and Metallurg, Eng, June 1946, pp. 101-103.

Hardy: Chem. Abst., vol. 45, March 1951, p. 2046a. 

1. A PROCESS FOR THE PREPARATION OF A PLASMA FRACTION FOR USE IN THE DETERMINATION OF THE PROTHROMBIN TIME OF THROMBOPLASTIC MATERIAL COMPRISING THE STEPS OF: DISSOLVING IN AXALATED PLASMS TWO TO TEN MILLIGRAMS PER MILLILITER OF OXALATED PLASMA TO A PLASMA FRACTION PREPARED FROM PROTHROMBIN-FREE PLASMA, SELECTED FROM THE CLASS CONSISTING OF HORSE, BOVINE, HUMAN, AND RABBIT PROTHROMOBIN-FREE PLASMA, BY A PROCESS CONDUCTED AT A TEMPERATURE WITHIN THE RANGE OF -5* TO 0*C. THROUGHOUT COMPRISING ADDING THERETO THREE TO FIVE VOLUMES OF A FIRST AQUEOUS ALCOHOL SOLUTION BUFFERED AT A PH OF 4-5 AND CONTAINING ABOUT 15-30 PERCENT BY VOLUME OF AN ALCOHOL SELECTED FROM THE CLASS CONSISTING OF METHANOL AND ETHANOL AND MIXTURES THEREOF, STIRRING CENTRIFUGING THE MIXTURE, SEPARATING THE SUPERNATANT FROM THE RESIDUE; AND ADDING TO THE RESIDUE THREE TO FIVE VOLUMES OF A SECOND AQUEOUS ALCOHOL SOLUTION BUFFERED AT A PH OF 6-8 AND CONTAINING 5-20 PERCENT BY VOLUME OF AN ALCOHOL SELECTED FROM THE CLASS CONSISTING OF METHANOL AND ETHANOL AND MIXTURES THEREOF AND 4-6 PERCENT BY WEIGHT OF AN ALKALI METAL SALT OF AN AMINO ACID HAVING NOT MORE THAN SIX CARBON ATOMS STIRRING AND CENTRIFUGING THE MIXTURE, FILTERING THE SUPERNATANT, DIALYZING THE FILTERED SUPERNATANT AGAINST DISTILLED WATER, AND LYOPHILIZING THE DIALYZED SUPERNATANT. 